Ergothioneine Stimulates Ca²⁺-Mediated Brain-Derived Neurotrophic Factor Expression in NE-4C Nerve Cells

Abstract

Ergothioneine (EGT), a naturally occurring histidine derivative, has been reported to modulate neurodegenerative diseases; however, the underlying mechanism remains unclear. This study aimed to investigate the brain-beneficial role of the natural amino acid EGT in NE-4C nerve cells. In the nerve cells, EGT treatment of >10 μM for 48 h significantly increased the expression of brain-derived neurotrophic factor (BDNF), as well as the phosphorylation of cAMP response element-binding protein (CREB), whereas no change was observed in acetylcholine receptor expression. Additionally, EGT induced an increase in intracellular Ca²⁺ levels via stimulation of the inositol 1,4,5-triphosphate receptor (IP₃R) in the endoplasmic reticulum; this increase was abrogated by the inhibition of organic cation transporter 1 (OCTN1). Structure–activity relationship analysis revealed the importance of the trimethylammonium group in EGT for intracellular events. In conclusion, EGT incorporated into cells via the OCTN1 route may act as a nerve transmission stimulator via IP₃R-mediated Ca²⁺-CREB/BDNF activation.

1. Introduction

Neurodegenerative diseases (NDDs), such as Alzheimer’s disease (AD) and Parkinson’s disease, are classified as neurological disorders characterized by the progressive loss of neurons in the central or peripheral nervous system.¹ Brain-derived neurotrophic factor (BDNF), a neurotrophin, has generated the most research interest due to its therapeutic potential for NDDs² because BDNF can ameliorate neuronal damage and promoted autophagy in SH-SY5Y cells³ and BDNF mRNA expression was lower in the brain of AD patients, compared to individuals with no AD.⁴ Therefore, the amelioration of the degraded BDNF signaling cascade is an alternative and appropriate strategy for the treatment or prevention of AD via diet because food compounds such as sulforaphane,⁵ polyphenols,⁶ peptides,⁷ and fish oil⁸ improved impaired cognition in animal studies mediated by BDNF activation.

Ergothioneine (EGT) is a naturally occurring sulfur-containing amino acid derived from histidine (His),⁹ commonly found in mushrooms. For example, Boletus edulis and yellow oyster mushrooms contain EGT at 7.27 mg/g and 7.18 mg/g, respectively.¹⁰ To date, EGT has attracted an increasing research attention owing to its physiological potential, including antioxidation,¹¹ antidiabetes,¹² and cardiovascular-protection,¹³ in human studies. In addition to these health benefits, Song et al.¹⁴ clarified that EGT intake in d-galactose-treated C57BL/6J mice activates the acetylcholine (ACh) nerve signaling pathways. Other reports also claimed the brain-health benefits of EGT with antiaging effect¹⁵ by increasing synapsin I expression in hippocampal cells¹⁶ and by eliminating senescent neuronal cells in hippocampus cells.¹⁷ The beneficial effect of EGT on the brain can be attributed to intact transport across the blood–brain barrier (BBB) and accumulation in the mouse brain (10.66 ng/mg-brain¹⁸), possibly via organic cation transporter 1 (OCTN1).¹⁹ In amyloid β (Aβ)_1–40-induced mouse models, brain-accessible EGT has been shown to prevent Aβ accumulation in the hippocampus by reducing acetylcholinesterase (AChE) activity.²⁰ However, the underlying mechanism of brain-beneficial EGT in the nervous system remains unclear.

In the present study, we explored the potential neurotransmission mechanisms of EGT in NE-4C nerve cells that were derived from the cerebral vesicles of a p53 gene-deficient mouse embryo²¹ because the cells possess ACh and BDNF nerve signaling systems targeted in this study.²² The structure–activity relationship was also analyzed using EGT and the analogues. In this study, we used l-hercynine (ERY), His, and 2-mercapto-l-histidine (mer-His), as depicted in Figure 1. Although EGT has two tautomeric thiol and thioketone forms, the predominant EGT under physiological situation is a thioketone form (Figure 1).²³ ERY was selected because of the lack of a thioketone group in the EGT structure. Mer-His was also selected because of the lack of the trimethylammonium moiety in the EGT structure, even though the thioketone group in EGT was replaced with the thiol group.

Figure 1.

Structures of ergothioneine (EGT) and its analogues. (l-hercynine, ERY; l-histidine, His; and 2-mercapto-l-histidine, mer-His.

2. Materials and Methods

2.1. Chemicals and Reagents

Eagle’s minimal essential medium (E-MEM) and retinoic acid (RA; Lot: SKH3179) were obtained from FUJIFILM Wako Pure Chemical Co. (Osaka, Japan). Fetal bovine serum (FBS) was purchased from Corning (Glendale, AZ, USA). l-EGT (Lot: 5-NSR-167-2) was purchased from Toronto Research Chemicals, Inc. (Toronto, Ontario, Canada). Poly-l-lysine solution (PLL; Lot: RNBM2127), ERY (Lot: 0000219037), mer-His (Lot: B02860979), 2-aminoethyl diphenylborinate (2-APB; Lot: BCBT7914), and pyrilamine maleate salt (Lot: MKCS9706) were purchased from Sigma-Aldrich (St. Louis, MO, USA). His (Lot: M8T1362) was purchased from Nacalai Tesque Co. (Kyoto, Japan). Dantrolene sodium (Lot: S547802) was purchased from Selleck Chemical Co. (Tokyo, Japan). An intracellular Ca²⁺ determination kit (Calcium Kit II-Fluo 4; Lot: WQ064) was purchased from DOJINDO Laboratories (Kumamoto, Japan).

2.2. Cell Culture

The neural stem cell line NE-4C (CRL-2925, Lot: 70050986) was purchased from the American Type Culture Collection (Manassas, VA, USA). The stem cells were cultured in PLL-coated 75 cm² flasks in E-MEM medium (containing l-glutamine, phenol red, sodium pyruvate, nonessential amino acids, and 1500 mg/L sodium bicarbonate) supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C in a 95% air/5% CO₂ humidified incubator. The cells were dissociated using 0.05% trypsin and transferred to new flasks when the growth confluence reached to 80–90%. Fifth-passage cells were used for all of the experiments.

2.3. Preparation of NE-4C Cell Lysate for Protein Expression Assay

NE-4C stem cells (9 × 10⁴ cells) were seeded in 35 mm PLL-coated dishes, followed by the addition of 1 μM RA in E-MEM containing 5% FBS for stem cell differentiation.²² After 4 days incubation, the nerve cells were rinsed twice with warm phosphate-buffered saline solution and lysed with ice-cold 1× radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris–HCl, 150 mM NaCl, 0.5% deoxycholic acid sodium salt, 0.1% sodium dodecyl sulfate, and 1% NP-40, pH 8.0) containing a protease (Nacalai Tesque Co.) and phosphatase (PhosSTOP, Roche, Basel, Switzerland) inhibitor cocktail tablets. After scraping, the obtained cell lysates were sonicated using Branson Digital Sonifier SFX 250 (Emerson Japan Co., Kanagawa, Japan) with an output control of 3 for 30 s at 4 °C, followed by centrifugation at 15,000g for 5 min at 4 °C (KUBOTA 3520, KUBOTA Co., Tokyo, Japan). An aliquot of the supernatant was used to determine the total protein concentration using the Pierce Protein Assay Kit (Lot: YG372899; Thermo Fisher Scientific, Waltham, MA, USA). The remaining supernatant was used for protein expression analysis via a Wes assay.

2.4. Measurement of Protein Expression by Wes

Protein expression levels were measured using a capillary electrophoresis-based immunoassay Wes instrument (ProteinSimple Co., San Jose, CA, USA) according to the manufacturer’s instructions. Briefly, the supernatant was diluted to 0.5 mg/mL with 0.1× sample buffer and 5× fluorescent master mix denaturing buffer, followed by denaturation at 95 °C for 5 min using the PCR Thermal Cycler (Takara Bio Inc., Shiga, Japan). After denaturation, a sample solution, biotinylated Wes reagents, and primary antibodies were loaded onto a microplate, followed by centrifugation (TOMY AX-511, TOMY Digital Biology Co., Tokyo, Japan) at 2500 rpm for 5 min at 25 °C. Wes measurements were performed using a 12–230 kDa separation module (8 × 25 mm capillary cartridge, ProteinSimple Co.). Automatic immunodetection was performed by using a horseradish-peroxidase-conjugated secondary antibody and a chemiluminescent substrate. Total protein was detected by attaching a pentafluorophenyl ester-biotin labeling reagent to the applied proteins. The operating conditions of the Wes instrument were as follows: separation time, 28 min; separation voltage, 375 V; antibody dilution time, 30 min; primary antibody time, 60 min; and secondary antibody time, 30 min. The chemiluminescent signal was displayed as a virtual blot-like image or electropherogram using the Compass for SW software (ProteinSimple Co.). Protein expression was normalized to the electropherogram peak area of the corresponding total protein in each lane, and the data are expressed as a ratio to the control group. The primary antibodies used for immunoblotting were as follows: BDNF (anti-BDNF antibody, rabbit monoclonal antibody, Lot: 1035294-1, 1:50 dilution, Abcam, Cambridge, UK), phospho-cAMP response element-binding protein (CREB) {pCREB (Ser133) [1B6], mouse monoclonal antibody, Lot: 11, 1:250 dilution, Cell Signaling Technology, Danvers, MA, USA}, CREB (anti-CREB antibody [D76D11], rabbit monoclonal antibody, Lot: 7, 1:50 dilution, Cell Signaling Technology), muscarinic AChR (anti-mAChR M1 antibody, Lot: 822203059, 1:50 dilution; GeneTex, CA, USA), and nicotinic AChR (anti-nAChR α4/CHRNA4 antibody, Lot: GR83312-9, 1:50 dilution; Abcam).

2.5. Measurement of Intracellular Ca²⁺ Levels in NE-4C Cells

The intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured using a Flex Station 3 (Molecular Devices Co., San Jose, CA, USA) according to the manufacturer’s instructions. Briefly, NE-4C stem cells seeded at a density of 3000 cells/well in a PLL-coated 96-well plate were used for this study. After differentiation with 1 μM RA, NE-4C nerve cells were incubated with a Fluo-4 AM loading buffer (100 μL/well, DOJINDO Laboratories) for 1 h at 37 °C in the dark. The loading buffer was formulated as follows: quenching buffer, Hanks’ HEPES Buffer, 5% Pluronic F-127, 250 mM probenecid, and 0.9 mM Fluo-4 AM. After recording the basal fluorescence intensity (F₀) for 30 s, the sample solution containing EGT, ERY, His, or mer-His (20 μL) was added to each well through a multichannel pipettor of Flex Station 3 and the fluorescence intensity (arbitrary unit) was measured for 90 s to record the maximum fluorescence (F_max) signal. Change in relative Fluo-4 fluorescence or change in [Ca²⁺]_i (RFU, relative fluorescence unit) was expressed as ΔF= (F_max – F₀). For inhibitor experiments, [Ca²⁺]_i levels in cells were measured by adding 100 μM EGT solution containing either 2-APB (an inhibitor of 1,4,5-triphosphate receptor [IP₃R],²⁴ 10 μM) or dantrolene (an inhibitor of ryanodine receptor [RyR],²⁵ 10 μM). To evaluate the involvement of transporters in EGT-induced [Ca²⁺]_i change, pyrilamine (an antagonist of OCTN1,²⁶ 0.5 mM) or His (a substrate for PHT1 (peptide/histidine receptor 1), 100 μM) was used for [Ca²⁺]_i experiments. After the baseline fluorescence (F₀) signal was recorded for 30 s, a sample solution was added to each well through a multichannel pipet included as a part of the fluidics module of Flex Station 3.

2.6. Statistical Analysis

Data are expressed as the mean ± standard deviation (SD) of distinct replicates. All analyses were performed using GraphPad Prism software (version 10.0, GraphPad; La Jolla, CA, USA). Statistical differences between multiple groups were evaluated using one-way analysis of variance (ANOVA), followed by Dunnett’s or Tukey’s post hoc test. p < 0.05 was considered as a statistically significant difference.

3. Results

3.1. Effect of EGT on BDNF Expression and CREB Phosphorylation in NE-4C Nerve Cells

A capillary electrophoresis-based immunoassay (Wes analysis) was performed to determine the effect of EGT on BDNF expression in NE-4C nerve cells. As shown in Figures 2A and S1, after a 2 day treatment of NE-4C progenitor cells with EGT, the expression of BDNF in the cells was significantly increased in a concentration-dependent manner (1–100 μM). Together with the increase in BDNF expression in NE-4C nerve cells, the expression of pCREB, an upstream signaling factor for BDNF generation,²⁷ was also increased by EGT (Figure 2B). This clearly suggested that EGT has physiological potential in stimulating CREB-mediated BDNF cascade in NE-4C nerve cells, similar to the upregulation of cascade by a food compound α-linolenic acid in PC12 cells.²⁸ Considering that m/n AChR expression was not affected by EGT (Figure 2C), the above finding may be due to an intracellular action of EGT within the cells.

Figure 2.

Effect of EGT on BDNF expression and CREB phosphorylation in NE-4C nerve cells. The expression of BDNF (A), ratio of pCREB and CREB (B), and m/n AChR (C) in NE-4C nerve cells treated with 1, 10, and 100 μM EGT were evaluated using Wes analysis. Protein expression of BDNF and m/n AChR was normalized using the electropherogram peak area of the total protein in each lane. The chemiluminescent signal was displayed as a virtual blot-like image, and an electropherogram was generated based on the molecular weight. Values are expressed as the mean ± SD (n = 3). Statistical analyses were performed using one-way ANOVA followed by Dunnett’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001 vs control; N.S., no significance at p > 0.05.

3.2. Effect of EGT on [Ca²⁺]_i in NE-4C Nerve Cells

In the EGT-induced CREB-BDNF signaling, the mechanism of signaling activation by EGT remains a mystery. Since West et al.²⁹ reported that an increasing [Ca²⁺]_i was a trigger for phosphorylation of CREB in neurons, further experiments in EGT-treated NE-4C nerve cells focused on the changes in [Ca²⁺]_i at different concentrations of EGT (1, 10, and 100 μM). As shown in Figure 3, in situ Fluo-4-fluorescence Ca²⁺ measurements revealed a rapid elevation in [Ca²⁺]_i after EGT addition in a concentration-dependent manner (1–100 μM). These findings strongly suggest that EGT directly increases intracellular Ca²⁺ levels via extracellular or intracellular events. Because of a significant (p < 0.0001 vs control) increase in [Ca²⁺]_i by 100 μM EGT, this concentration was used for further Ca²⁺ experiments.

Figure 3.

Effect of EGT on intracellular Ca²⁺ concentration ([Ca²⁺]_i) in NE-4C nerve cells. (A) Real-time change of intracellular fluorescence signals with time in NE-4C nerve cells. The stimulation of EGT (1, 10, and 100 μM) started after 30 s of monitoring basal fluorescence intensity. (B) Transient changes of fluorescence from Fluo-4 after stimulation of EGT (1, 10, and 100 μM) in NE-4C nerve cells. Calcium kit II—Fluo 4 was used in this study. Transient changes in [Ca²⁺]_i [relative fluorescence units ,(RFU)] were calculated as the difference between peak (F_max) and basal (F₀) fluorescence intensity (ΔF = F_max – F₀) at excitation wavelength of 485 nm, emission wavelength of 525 nm, and cutoff filter of 515 nm. RFU, relative fluorescence units. Values are expressed as the mean ± SD (n = 3 or 4). Statistical analyses were performed using one-way ANOVA followed by Dunnett’s t-test. *p < 0.05, ****p < 0.0001 vs control.

3.3. Structure–Activity Relationship between EGT and Increase in [Ca²⁺]_i in NE-4C Nerve Cells

To assess the structural factors responsible for the EGT-induced increase in [Ca²⁺]_i, EGT and its analogues (ERY, His, and mer-His) were subjected to [Ca²⁺]_i measurements in NE-4C nerve cells. As shown in Figure 4, EGT and ERY molecules, both of which have a trimethylammonium group, evoked a significant increase in [Ca²⁺]_i, whereas no significant changes in [Ca²⁺]_i were observed with His or mer-His molecules, both of which lack this group. Structure–activity relationship analysis also revealed that the thioketone group in the imidazole moiety did not contribute to the observed increase in [Ca²⁺]_i by EGT (Figure 3). CREB phosphorylation and BDNF expression were also stimulated by EGT and ERY but not by His and mer-His (Figure S2), indicating that the trimethylammonium group is a key structural factor for the activation of the BDNF signaling cascade in NE-4C nerve cells.

Figure 4.

Structure–activity relationship achieved by contrasting the effect of EGT and its analogues on [Ca²⁺]_i in NE-4C nerve cells. (A) Real-time change of intracellular fluorescence signals with time in NE-4C nerve cells. The stimulation of sample (EGT, ERY, His, and mer-His) started after 30 s of monitoring basal fluorescence intensity. The concentration of each sample is 100 μM. (B) Transient changes of the intracellular fluorescence after sample stimulation in NE-4C nerve cells. Values are expressed as the mean ± SD (n = 4). Statistical analyses were performed using one-way ANOVA followed by Tukey–Kramer’s t-test among multiple groups. Different letters represent the statistical difference at p < 0.05.

3.4. Transport Route of EGT in NE-4C Nerve Cells

The transport routes of EGT in NE-4C nerve cells were investigated to clarify the increase in [Ca²⁺]_i via extracellular or intracellular events as a function of [Ca²⁺]_i, using pyrilamine, an OCTN1 antagonist, and His, a substrate for PHT1. As shown in Figure 5, pyrilamine, but not His, significantly blocked the [Ca²⁺]_i elevation induced by EGT and ERY. This strongly suggests that the elevation of [Ca²⁺]_i by EGT (Figure 3), following the activation of CREB/BDNF signaling (Figure 2A,B), may be due to intracellular EGT being incorporated into NE-4C nerve cells via the OCTN1 transport route. This is consistent with a previous report indicating that EGT was incorporated into human embryonic kidney 293 cells through the OCTN1 transport route.³⁰

Figure 5.

Involvement of OCTN1 and PHT1 in EGT-or ERY-induced Ca²⁺ release in NE-4C nerve cells. (A) Changes in [Ca²⁺]_i in NE-4C nerve cells treated with EGT or ERY (100 μM) in the presence or absence of pyrilamine (antagonist of OCTN1, 0.5 mM). (B) Alternations of [Ca²⁺]_i in NE-4C nerve cells treated with EGT or ERY (100 μM) with or without His (substrate of PHT1, 100 μM). After 30 s of monitoring basal fluorescence intensity, the prepared samples were added to NE-4C nerve cells. Values are expressed as the mean ± SD (n = 4). Statistical analyses were performed using one-way ANOVA followed by Tukey–Kramer’s t-test among multiple groups. Different letters represent the statistical difference at p < 0.05. OCTN1, organic cation transporter 1; PHT1, peptide/histidine transporter 1.

3.5. Mechanism of EGT-Induced Increase in [Ca²⁺]_i in NE-4C Nerve Cells

Based on the above finding that the increase in [Ca²⁺]_i resulted from the incorporation of EGT into the cells, we focused on the release of Ca²⁺ from the endoplasmic reticulum (ER), which serves as a main dynamic Ca²⁺ storage organelle in cells.³¹ When the Ca²⁺ released from the ER was blocked using inhibitors for IP₃R and RyR, which are intracellular Ca²⁺ channels,³² inhibition of IP₃R by 2-APB significantly abolished EGT-induced [Ca²⁺]_i elevation; however, no changes were observed in [Ca²⁺]_i in EGT-treated NE-4C cells due to RyR inhibitor dantrolene (Figure 6). These results suggest that EGT and ERY molecules bearing a trimethylammonium group may act as IP₃R-specific stimulators or Ca²⁺ release promoters, leading to activation of the CREB/BDNF signaling pathway.

Figure 6.

Effect of intracellular Ca²⁺ release channel on EGT-induced increase in [Ca²⁺]_i in NE-4C nerve cells. (A) Effect of 2-APB (inhibitor of IP₃R) on EGT-stimulated [Ca²⁺]_i changes in NE-4C nerve cells. (B) Impact of 2-APB on ERY-induced [Ca²⁺]_i increase in nerve cells. (C) Effect of dantrolene (inhibitor of RyR) on EGT-stimulated [Ca²⁺]_i upregulation in nerve cells. After 30 s of monitoring basal fluorescence intensity, the prepared samples were added to NE-4C nerve cells. Values are expressed as the mean ± SD (n = 3 or 4). Statistical analyses were performed using one-way ANOVA followed by Tukey–Kramer’s t-test among multiple groups. Different letters represent the statistical difference at p < 0.05. 2-APB, 2-aminoethoxydiphenyl borate; IP₃R, inositol 1,4,5-trisphosphate receptor; RyR, ryanodine receptor. 2-APB, 10 μM; EGT, 100 μM; ERY, 100 μM; and dantrolene, 10 μM.

4. Discussion

EGT, a natural amino acid derived from His, has been shown to exhibit a variety of physiological functions, including NDD prevention effect, in animals and humans.^{11−13,20,33−35} Yang et al.²⁰ reported that the daily intake of EGT (2 mg/kg) in AD mice for 51 days ameliorated the Aβ_1–40-induced loss of memory and learning abilities by reduced Aβ accumulation in the hippocampus. Nakamichi et al.³⁵ also provided the evidence that the oral intake of EGT (50 mg/kg, 2 weeks) enhances object recognition memory in normal ICR mice via the promotion of neuronal maturation. However, the mechanism underlying these effects of EGT is not fully understood.

This study, for the first time, provided evidence that EGT activates the CREB/BDNF pathway in NE-4C nerve cells (Figure 2A,B). Similarly, food compounds, such as sulforaphane,⁵ α-linolenic acid,²⁸ lycopene,³⁶ and peptides,³⁷ have been reported as potential candidates for the upregulation of BDNF cascade. However, except for α-linolenic acid, which activated the protein kinase A (PKA)/CREB/BDNF signaling cascade in PC12 cells, the mechanism(s) underlying CREB/BDNF activation of the above food compounds remained unascertained. Considering the reported bioavailability¹⁸ and a long stability (half-life, 1 month in rats³⁸) of EGT, the intake would be a benefit for brain health, compared to the above neuroprotective compounds. BDNF is upregulated by the stimulation of transmembrane ACh receptors (m/n AChR) in the rat hippocampus and cerebral cortex.^39,40 However, in this study, EGT with a trimethylammonium group, such as ACh, did not affect m/n AChR expression (Figure 2C), suggesting that EGT-induced activation of the CREB/BDNF pathway (Figure 2) may be caused by an intracellular event and not AChR-mediated pathways.

For the stimulation of CREB/BDNF signaling, Ca²⁺ plays a crucial role in the activation of Ca²⁺/calmodulin-dependent kinase II (CaMKII), p38 mitogen-activated protein kinase (MAPK)-mediated BDNF cascade,⁴¹ and tyrosine receptor kinase B (TrkB)-mediated signaling pathway. Based on the present findings that the increased BDNF expression was due to the incorporation of EGT via the OCNT1 route, a reported EGT transport route³⁰ (Figure 5A), we further aimed to clarify whether EGT affects intracellular Ca²⁺ levels. As shown in Figure 3, EGT significantly increased [Ca²⁺]_i in NE-4C nerve cells. Zhong et al.⁴² reported that peoniflorin, a monoterpene glycoside isolated from herbal medicine, attenuated Aβ-induced neurotoxicity via maintenance of [Ca²⁺]_i homeostasis in the rat hippocampus. In addition, gastrodin, a bioactive compound derived from a Chinese herb, was also found to ameliorate memory impairment via modulation of the Ca²⁺/CaMKII pathway in rats with vascular dementia.⁴³ Although the involvement of TrkB or p38-MAPK for Ca²⁺-mediated CREB/BDNF activation remained unclear, intracellular Ca²⁺ elevation by the natural compounds including EGT may be a trigger for the activation of the CREB/BDNF cascade. To further investigate the structure–activity relationship (Figures 4 and S2) and [Ca²⁺]_i elevation experiments (Figure 6), we identified, for the first time, that the trimethylammonium group in EGT is a key structural component responsible for increasing [Ca²⁺]_i via IP₃R stimulation. This is in line with a report that trimethylamine N-oxide, a metabolic product of choline, enhances Ca²⁺ release from platelet stores by augmenting IP₃ signaling pathways.⁴⁴ Further studies are in progress to explore the interaction between the trimethylammonium group in EGT and the IP₃R protein in the ER using our previously reported CHARMM-GUI molecular docking analysis.⁴⁵

In the current study, even though we provided the first finding that EGT has potential for the activation of the Ca²⁺-CREB/BDNF signaling pathway in NE-4C nerve cells, the potential translation of these findings into in vivo animal and human models is an issue that cannot be ignored. Tang et al.¹⁸ studied the tissue distribution of EGT after oral administration in C57BL6J mice (70 mg/kg/day, 28 days) and found that the concentration of EGT reached 840 μM and 10.66 ng/mg-brain in the mouse blood and brain tissue, respectively. In addition, in human administration study of mushroom-extract tablet containing 5 mg of EGT for 12 weeks, the concentration was estimated to be 497 μM in blood.³³ Taken together, the current results obtained at >10 μM of EGT would be acceptable under physiological conditions, but bioavailability analysis of EGT is still needed for further study. Considering the limitations of the current NE-4C cell model, the EGT-stimulated neurotransmission will be further explored in other neuronal cell models, such as SH-SY5Y cells⁴⁶ and Neuro-2a cells,⁴⁷ and in animal models, such as senescence-accelerated mouse model.⁴⁸ Besides, the synergistic effect of EGT with neuroprotective agents is also a promising and innovative research direction.

In conclusion, we demonstrated that EGT, a His metabolite commonly found in mushrooms¹⁰ and Aspergillus oryzae-fermented rice bran,⁴⁹ stimulates neurotransmission via the IP₃R-mediated Ca²⁺-CREB/BDNF signaling pathway in NE-4C nerve cells (Figure 7). The structure–activity studies revealed that the presence of the trimethylammonium group in EGT is essential for activity, while the thioketone group does not affect the activity. Additionally, considering the high bioavailability of EGT, it would be a brain-beneficial food compound across the BBB, like Tyr–Pro,⁵⁰ after oral diet of EGT or EGT-containing foods.

Figure 7.

Schematic signaling pathway of EGT toward BDNF expression in NE-4C nerve cells.

Glossary

Abbreviations

ACh

acetylcholine

m/n AChR

muscarinic/nicotinic ACh receptor

2-APB

2-aminoethyl diphenylborinate

BBB

blood–brain barrier

AD

Alzheimer’s disease

BDNF

brain-derived neurotrophic factor

CaMKII

Ca²⁺/calmodulin-dependent kinase II

CREB

cAMP response element-binding protein

EGT

l-ergothioneine

E-MEM

Eagle’s minimal essential medium

ER

endoplasmic reticulum

ERY

l-hercynine

FBS

fetal bovine serum

IP₃R

inositol 1,4,5-triphosphate receptor

MAPK

mitogen-activated protein kinase

MCI

mild cognitive impairment

mer-His

2-mercapto-l-histidine

NDD

neurodegenerative disease

NGF

nerve growth factor

NT

neurotrophin

OCTN1

organic cation transporter 1

PBS

phosphate-buffered saline

PD

Parkinson’s disease

PHT1

peptide/histidine transporter 1

PKA

protein kinase A

PLL

poly l-lysine solution

RA

retinoic acid

RIPA

radioimmunoprecipitation assay

RyR

ryanodine receptor

RFU

relative fluorescence unit

TrkB

tyrosine receptor kinase B

Data Availability Statement

All data generated or analyzed during this study are included in this article and supplementary documents.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.4c09920.

• Uncropped virtual blot-like images of BDNF, pCREB/CREB, and m/n AChR using the Wes analysis; NE-4C progenitor cells treated with EGT (1, 10, and 100 μM) for 48 h; uncropped virtual blot-like images of pCREB/CREB and BDNF using the Wes analysis; NE-4C progenitor cells treated with EGT and its analogues (ERY, His, and mer-His) at a concentration of 100 μM for 48 h (PDF)

Author Contributions

C.S. and S.A. performed cell line experiments. C.S., S.A., S.N., and T.M. analyzed and discussed the results. C.S. drafted the original manuscript. S.N. and T.M. reviewed and edited the manuscript. T.M. designed and supervised the study. All authors have read and approved the final version of the manuscript for publication.

The authors declare no competing financial interest.